Activation of cannabinoid receptors in breast cancer cells improves osteoblast viability in cancer-bone interaction model while reducing breast cancer cell survival and migration

The endocannabinoid system has been postulated to help restrict cancer progression and maintain osteoblastic function during bone metastasis. Herein, the effects of cannabinoid receptor (CB) type 1 and 2 activation on breast cancer cell and osteoblast interaction were investigated by using ACEA and GW405833 as CB1 and CB2 agonists, respectively. Our results showed that breast cancer cell (MDA-MB-231)-derived conditioned media markedly decreased osteoblast-like UMR-106 cell viability. In contrast, media from MDA-MB-231 cells pre-treated with GW405833 improved UMR-106 cell viability. MDA-MB-231 cells were apparently more susceptible to both CB agonists than UMR-106 cells. Thereafter, we sought to answer the question as to how CB agonists reduced MDA-MB-231 cell virulence. Present data showed that co-activation of CB1 and CB2 exerted cytotoxic effects on MDA-MB-231 cells by increasing apoptotic cell death through suppression of the NF-κB signaling pathway in an ROS-independent mechanism. ACEA or GW405833 alone or in combination also inhibited MDA-MB-231 cell migration. Thus, it can be concluded that the endocannabinoid system is able to provide protection during breast cancer bone metastasis by interfering cancer and bone cell interaction as well as by the direct suppression of cancer cell growth and migration.

www.nature.com/scientificreports/ Thereafter, they were treated with ACEA, GW405833 or the combination of both for the indicated times. MTT (Invitrogen, CA, USA) solution was added to the final concentration of 0.5 mg/mL in 100 µL in serum-free DMEM and incubated at 37 °C for 4 h, then stop solution was added and thoroughly mixed. The optical density at 595 nm was measured using a microplate reader (Multiskan EX) (Thermo Fisher Scientific, MA, USA). The percentage of cell viability was calculated based on the absorbance ratio between cells treated with the compounds and control multiplied by 100 (percent cell viability normalized to control).
Apoptosis assay. MDA-MB-231 cells were seeded into a 6-well plate at a density of 3.3 × 10 5 cells/well and allowed to attach overnight. Thereafter, the cells were treated with ACEA, GW405833 or the combination of both for 48 h. At the end of the experiment, cells were stained with Annexin-V-FITC (A13199) (Invitrogen) and propidium iodide (PI) (P3566) (Invitrogen) according to the manufacturer's guideline. Apoptotic cells were quantified using FACScan flow cytometer (BD FACSCanto) (BD Biosciences, CA, USA). Cells were analyzed for the percentage of cells in healthy, early apoptotic, late apoptotic and necrotic phases by using FACSDiva™ Software version 6.1.3 (BD Biosciences).
DCFDA cellular ROS detection assay. Cellular    www.nature.com/scientificreports/ Western blot analysis. Twenty micrograms of protein samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Invitrogen) gel before being transferred to nitrocellulose blotting membrane (GE Healthcare, Texas, USA).The membranes were then blocked with 5% (w/v) bovine serum albumin (BSA) (Capricorn Scientific, Hessen, Germany) for 2 h at room temperature, followed by the incubation at 4 °C overnight with primary antibodies. After washing, the membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. Proteins on the immunoblots were visualized by enhanced chemiluminescence (ECL) (Millipore, MA, USA) and exposed to X-ray film (GE Healthcare). Protein band intensity was quantified using ImageJ software (National Institutes of Health, MD, USA). To relatively quantify the target protein expression, the intensity of target protein band was normalized to β-actin of each lane.
Cell migration assay. Cell migration was measured by wound healing assay modified from Lertsuwan et al., 27 . Briefly, MDA-MB-231 cells were seeded into a 24-well plate at a density of 4.0 × 10 5 cells/well and allowed to attach overnight. Thereafter, wound was created with a sterile 200-µL pipette tip, then the cells were treated with ACEA, GW405833 or the combination of both. Series of images of the scratched wound were taken under a phase contrast inverted microscope (model AE31) (Motic, China) from 0 to 36 h. The closure of the wound representing migration ability of the cells was measured by using ImageJ software (National Institutes of Health). Cell migration was calculated as the percentage of wound closure area compared to the initial scratched area.

Statistical analysis.
All results are expressed as means ± standard errors (SEM). Unless otherwise specified, statistical analysis for multiple comparisons was performed using one-way analysis of variance (ANOVA). The difference between pairs of means was analyzed by Tukey post-test. A p-value < 0.05 was considered statistically significant. Calculation of the inhibitory concentration at 50% (IC 50 ) and all statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc., USA).

MDA-MB-231 cell-derived CM inhibited osteoblast-like UMR-106 cell survival, while pretreatment of GW405833 prevented this effect. Since breast cancer cells appear to have a great propensity
for bone metastasis, the ability of breast cancer cells to alter osteoblast viability was determined by indirect-contact experiment. We evaluated the indirect effect of MDA-MB-231 cell-derived CM on osteoblast-like UMR-106 cell survival using MTT assay (Fig. 1a). As shown in Fig. 1b, UMR-106 cell viability was significantly inhibited by 46.75% when cells were treated with fifty percent of MDA-MB-231 cell-derived CM as compared to those treated with fifty percent serum free media. To further elaborate the potential effects of CB1 and CB2 agonists on this interaction, CM collected from MDA-MB-231 cell-pretreated with ACEA, GW405833 or their combination were used. While pretreatment with ACEA or the combination did not alter CM-mediated osteoblast suppression, significant recovery was observed in UMR-106 cells treated with CM from MDA-MB-231 pretreated with CB2 agonist, GW405833 (Fig. 1c). It was thus suggested that the ECS-particularly neoplastic CB2 activationwas able to alleviate the negative effect of breast cancer cells on osteoblasts.  Tables 1 and 2). Specifically, UMR-106 exhibited markedly higher IC 50 values reflecting its lower sensitivity to both ACEA and GW405833 than MDA-MB-231 (Table S1) (Tables 1 and 2). On average, MDA-MB-231 cells were more sensitive to ACEA and GW405833 than UMR-106 cells by 1.44-fold and 6.76-fold, respectively (Table S1).

Combination of ACEA and GW405833 augmented their cytotoxicity in MDA-MB-231 cells.
As seen in Fig. 3, exposure to specific CB1 or CB2 agonist alone significantly inhibited MDA-MB-231 cell viability. We therefore evaluated whether the co-activation of CB1 and CB2 receptors by ACEA and GW405833 at different combination ratio could enhance their cytotoxic effects. As GW405833 was apparently more toxic to MDA-MB-231 than ACEA ( Fig. 3 and Table 2   www.nature.com/scientificreports/  www.nature.com/scientificreports/   www.nature.com/scientificreports/ MDA-MB-231 cells (Fig. 5a,b,c,d,e,f). Our results revealed that the percentage of normal cells was significantly decreased in the combined treatment as compared to other groups (Fig. 5b). This corresponded to a significant increase in early apoptotic, late apoptotic and total apoptotic cell proportion in MDA-MB-231 cells (Fig. 5d,e,f). On the other hand, the cells in necrotic phase in all treated groups remained unchanged as compared to the control (Fig. 5c). The finding indicated that the combination of CB1 and CB2 agonists at the ratio of 2:1 led to apoptosis of MDA-MB-231 cells rather than necrosis.
Combination of ACEA and GW405833 suppressed nuclear factor-κB (NF-κB) signaling pathway in MDA-MB-231 cells. Since ROS plays an important role in apoptosis in many cell types 28,29 , we then examined whether cellular ROS level was altered in MDA-MB-231 after exposing to ACEA, GW405833 or their combination. It was shown that cellular ROS levels in all treated cells remained unchanged as compared to control group (Fig. 6a). Furthermore, we examined whether the combined treatment of ACEA and GW405833 affected the apoptosis-associated proteins. Our data showed that the expression of effector caspase-3 was significantly increased in the combined treatment ( Fig. 6b and Fig. S1). Because NF-κB is often involved in the regulation of several signaling pathways including apoptosis, we further investigated whether the combined treatment affected the expression of p-NF-κB p65. It was apparent that while the level of p-NF-κB p65 remained unchanged in individual treatments, p-NF-κB p65 was significantly reduced in the combined treatment group by 41.57% (Fig. 6c and Fig. S1).

ACEA and GW405833 inhibited MDA-MB-231 cell migration.
Since migration is one of the important characteristics of aggressive cancer and also plays an important role during cancer bone metastasis 30-32 , we further examined whether ACEA and GW405833 could suppress MDA-MB-231 cell migration using wound healing assay. The results showed that the percentage of wound closure, which represented cell migration, was significantly decreased in the presence of 20 µM ACEA at 24 h and 30 µM ACEA at 24 and 36 h (Fig. 7a,b). Similarly, significant cell migration suppression was found in MDA-MB-231 cells treated with 15 µM GW405833 at 36 h (Fig. 7c,d).
As the combined treatment markedly reduced cancer cell viability, we then explored whether the combination of ACEA and GW405833 could further suppress MDA-MB-213 cell migration. This series of experiments confirmed a significant reduction in MDA-MB-231 cell migration by 30 µM ACEA or 15 µM GW405833 given alone at 24 h, whereas a combination of both agonists did not synergistically enhance the inhibitory effects on MDA-MB-231 cell migration (Fig. 8a,b).

Discussion
ECS has been shown to modulate cancer progression and bone homeostasis. CB expression switch was reported in cancer progression and was probably associated with poor prognosis and cancer aggressiveness 15 . Meanwhile, both CB1 and CB2 were crucial for bone homeostasis 33 since they could regulate bone formation and resorption. Activation of CB2 promoted osteoblast cell survival and favored bone formation 34-36 while inhibiting osteoclast differentiation and function-especially in postmenopausal osteoporosis, thus correlating with positive effect of estrogen on CB2 expression in both human and animal model 37 . Mice with CB2 deficiency exhibited a significant reduction in bone mass and develop osteoporosis reflecting crucial roles of CB2 in bone homeostasis. On the other hand, stimulation of CB1 with synthetic agonist resulted in increased osteoclast differentiation and bone resorption activity 36,38 . Nevertheless, CB1 deficiency mice was shown to have increased bone mass only at their early developmental stage (less than 3 months), but they later developed age-related osteoporosis with high level of lipid accumulation in bone. MSCs (bone marrow stromal cells) from these mice had lower osteogenic differentiation but higher adipogenic differentiation ability. Therefore, CB1 was hypothesized to function in MSC differentiation toward osteogenic linage as well 37 . Accordingly, ECS was involved in the regulation of bone remodeling, and the presence of both CB1 and CB2 was important for maintaining bone homeostasis.
Interaction between cancer and bone cells appeared to be crucial during cancer bone metastasis when cancer cells regulated bone cell differentiation and activity to facilitate their colonization in bone. This phenomenon was also evident in breast cancer when breast cancer cells secreted a number of factors, such as transforming growth factor-β 1 (TGF-β 1 ), interleukin 1 (IL-1), IL-6 and IL-11 14,39-41 , all of which suppressed osteoblast differentiation and function. Previous study also reported that CM collected from breast cancer cells induced osteoblast cell death 13 , thus consistent with our finding that breast cancer MDA-MB-231 cell-derived CM inhibited osteoblastlike UMR-106 cell viability. Besides, conditioned media from metastatic breast cancer cell also reportedly induced the expression and secretion of inflammatory cytokines including interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1 in osteoblasts. Since these factors were known to attract osteoclast precursor cells and promote osteoclast differentiation, this mechanism could increase bone resorption during breast cancer bone metastasis 42,43 . Interestingly, CB2 agonist (GW405833) pretreatment prevented the toxic effects of MDA-MB-231 cell-derived CM on UMR-106 cells. This result corresponded to the inhibitory effects of CB2 agonists on breast cancer bone colonization and cancer-induced bone loss reported in rodents 44,45 . Our results further showed that CB agonist treatments of breast cancer cells also reduced the activation of NF-κB, which were involved in cancer-bone interaction, presumably by regulating the expression of inflammatory cytokines in breast cancer (as reviewed in Gordon et al.) 46 .
In addition to the positive roles of ECS in bone formation as mentioned previously, our data also suggested another aspect of how ECS could protect bone microenvironment during breast cancer bone metastasis by interfering with cancer and bone interaction, particularly by counteracting the negative effects of cancer cells www.nature.com/scientificreports/  www.nature.com/scientificreports/ on osteoblast viability. Other studies also showed that CB2 activation in osteoblasts upregulated the production of RANKL and OPG, cytokines functioning in osteoclast-osteoblast crosstalk 35 . Thus, CB2 agonist in bone microenvironment could facilitate bone formation and compromising the interaction between cancer cells versus osteoclast and/or osteoclasts at the same time. Nevertheless, more studies are required to uncover the mechanism by which ECS prevents the breast cancer cell-mediated osteoblast suppression. Previous studies reported that the activation of CB1 or CB2 alone suppressed tumor growth and induced cancer cell death 16,17 . Since both CB1 and CB2 ligands were present in bone microenvironment 5,47 , breast cancer cell responses to different combination of CB1 and CB2 agonists were investigated in this study. Both ACEA (CB1 agonist) and GW405833 (CB2 agonist) were shown to suppress the viability of both MDA-MB-231 and UMR-106 cells with MDA-MB-231 cells being more sensitive to ACEA and GW405833 than UMR-106 cells. Interestingly, the growth stimulatory effect of low concentrations of ACEA and GW405833 on osteoblasts was also observed. The expression of CB1 and CB2 on the cell membrane of MDA-MB-231 and UMR-106 cells has previously been demonstrated 38,48 . However, there has been no comparative study on the relative distribution of CB1 and CB2 on the two cell types. With the opposite effects, i.e., growth promoting effect of low concentration of CB agonists on breast cancer and osteoblast cell growth, we hypothesized that the differential responses of MDA-MB-231 and UMR-106 cells to CB agonists was likely to result from different downstream signaling pathways. www.nature.com/scientificreports/ Indeed, several cell types in bone microenvironment, e.g., endothelial cells, basophils and macrophages, are able to produce endocannabinoids 49,50 . It is noteworthy that immune response is the very first response when cancer cells metastasize to a new environment including bone 51 . Since cancer cells extravasate from blood vessels to the sinusoidal area of bone, they would be exposed to the endocannabinoid-rich environment in the highly vascularized area of the bone. CB activation has been reported to facilitate immune cell proliferation and activity 50 . Moreover, previous study also showed that the production of endocannabinoids was significantly increased in the activated lymphocytes as compared to the inactivated cells 52 . Therefore, immune activation from cancer metastasis could further upregulate the endocannabinoids production from immune cells at the metastatic site. Taken together, the arriving cancer cells would presumably be exposed to high concentrations of endocannabinoid ligands released from the endothelial and immune cells in the sinusoidal milieu. In other words, only the arriving cancer cells, but not distant osteoblasts, would be compromised by the high concentrations of CB agonists.
Furthermore, potential mechanism underlying the negative effects of CB agonists on breast cancer cell survival was investigated in MDA-MB-231 exposed to each CB agonist alone and the combined treatment. Our findings suggested that simultaneous exposure to both agonists in bone microenvironment might provide stronger protective effects during breast cancer bone metastasis. Our data corresponded to the synergistic effects of CB1 and CB2 agonists on breast cancer growth inhibition and tumor-induced pain suppression reported previously 53,54 . Our results showed that simultaneous activation of CB1 and CB2 enhanced cancer cell death via apoptosis rather than necrosis, and the mechanism involved downregulation of the phosphorylated NF-κB.  www.nature.com/scientificreports/ Apoptotic cell death is often followed by effective cell clearance, which prevents the release of cell content and inflammation within bone environment 55 ; therefore, this process should not induce osteoclastogenesis due to proinflammatory cytokine production. Previous studies showed that activation of CB1 and CB2 in breast cancer suppressed ERK1/2 and AKT/mTOR signaling pathways [18][19][20] . On the other hand, NF-κB, one of the downstream effectors of both pathways, was shown to be associated with cancer cell survival and progression [21][22][23] . Inhibition of NF-κB activity in breast cancer was also shown to induce apoptosis corresponding to the reduced phosphorylated NF-κB level and enhanced apoptosis in MDA-MB-231 cells treated with CB agonists in this study 56 . Taken together, this study strongly suggested the role of NF-κB pathway in the CB-mediated cancer cell suppression. In addition, the involvement of ROS production in cancer cell progression and apoptosis was reported 24 . On the other hand, ROS has been known to be associated with several bone diseases by promoting osteoclast activities while suppressing osteoblast function 25 . However, we found that ROS production was not increased in CB agonist-exposed MDA-MB-231 cells in this study. In other words, ECS was capable of inducing breast cancer cell apoptosis in a pathway that did not involve ROS and its harmful effect from ROS on bone homeostasis. Since cell migration was markedly inhibited by CB agonists, it was likely that ECS may also help restrict the spreading of breast cancer cells within bone microenvironment.
In conclusions, we have demonstrated that the ECS-which was present in bone microenvironment-provided a protection against breast cancer bone metastasis and its negative consequence on bone cell survival. Specifically, CB agonists, especially CB2 agonist, was able to prevent breast cancer-induced osteoblast suppression. Each of the two CB agonists or a combination of both could reduce breast cancer cell survival and migration through the NF-κB-dependent pathway. Regarding the limitation of the present study, we realize that a series of studies on the plasma membrane expression levels and binding affinity of CB1 and CB2 receptors might help www.nature.com/scientificreports/ explain the differential responses of MDA-MB-231 cells vs. osteoblasts to CB agonists. Although additional studies are also needed to confirm the present findings in an in vivo model, we have provided evidence to support the novel roles of ECS in breast cancer bone metastasis, thus promoting better understanding of the pathophysiological importance of ECS within bone microenvironment. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.